Development in all living organisms is controlled by the precise arrangement of molecular cues. For proper development, these cues must be present at specific concentrations and in defined locations. Traditional biological approaches to studying development make use of knockout animals, which harbor single or multiple gene deletions;however, these gene deletions can simultaneously affect numerous developmental events. We seek to develop novel chemical tools to examine specific developmental events. This will be accomplished through the advancement of new surface chemistry, capable of interweaving multiple molecules with defined concentration gradients on a single substrate. Here, we describe the application of this chemistry to problems in neuronal wiring, however it is broadly applicable to many problems in biology and materials. Moreover, the complex developmental models described may serve as novel starting points for in vitro drug discovery. The proposed program of study will provide a new tool kit for examining a wide array of biological problems that are not accessible using classical biological techniques. In the project description, we describe both the development of this tool kit and its application to problems in neuronal wiring. We seek to unravel fundamental mysteries in development by recreating complex developmental patterns in vitro. As an initial system, we have chosen to examine midline crossing of zebrafish (Danio rerio) optical neurons. This problem is an attractive starting point, since it has been extensively studied using classical methods. While classical analysis has not unambiguously elucidated this wiring pathway, these studies provide a clear starting point for our models. Long-term, the complex developmental maps produced using these techniques have the potential to provide intricate in vitro models for developmental defects, tissue damage, and drug discovery. PUBLIC HEALTH RELEVANCE: The proposed program of study will develop new methods for understanding how neuronal connections are formed in the brain. This methodology will lead to a better understanding of developmental neuronal disorders in humans. Additionally, the complex models developed for this project are a potential basis for drug discovery.